Keap1 facilitates p62-mediated ubiquitin aggregate clearance via autophagy

Abstract

The accumulation of ubiquitin-positive protein aggregates has been implicated in the pathogenesis of neurodegenerative diseases, heart disease and diabetes. Emerging evidence indicates that the autophagy lysosomal pathway plays a critical role in the clearance of ubiquitin aggregates, a process that is mediated by the ubiquitin binding protein p62. In addition to binding ubiquitin, p62 also interacts with LC3 and transports ubiquitin conjugates to autophagosomes for degradation. The exact regulatory mechanism of this process is still largely unknown. Here we report the identification of Keap1 as a binding partner for p62 and LC3. Keap1 inhibits Nrf2 by sequestering it in the cytosol and preventing its translocation to the nucleus and activation of genes involved in the oxidative stress response. In this study, we found that Keap1 interacts with p62 and LC3 in a stress-inducible manner, and that Keap1 colocalizes with LC3 and p62 in puromycin-induced ubiquitin aggregates. Moreover, p62 serves as a bridge between Keap1 and ubiquitin aggregates and autophagosomes. Finally, genetic ablation of Keap1 leads to the accumulation of ubiquitin aggregates, increased cytotoxicity of misfolded protein aggregates, and defective activation of autophagy. Therefore, this study assigns a novel positive role of Keap1 in upregulating p62-mediated autophagic clearance of ubiquitin aggregates.

Degradation of Keap1 activates BH3-only proteins Bim and PUMA during hepatocyte lipoapoptosis

“…39 Also, Cathepsin B, a major lysosomal cysteine protease implicated in FFA-induced mitochondrial dysfunctions, 40 is overexpressed in beta cells during excessive autophagy. 41 In accord with a previous report, 27 our study reveals that depletion of Keap1 induces cell death in liver cell lines by activating Bim and PUMA via JNK (vide infra) and contributes to saturated FFA-induced toxicity. Thus Keap1 degradation could represent an additional mechanism by which autophagy regulates cell death.…”

“…Recent studies indicate that Keap1 can bind to the autophagy substrate Sequestrosome (SQSTM)1/p62, resulting in Keap1 degradation through autophagy. 26,27 Therefore we sought to determine whether PA stimulates autophagy. PA treatment increased protein levels of the lipidated form of LC3 (LC3-II) in Hep3B ( Figure 2b) and Huh-7 cells (Supplementary Figure S1A), an indicator of autophagosome formation.…”

“…Consistent with these latter observations, several studies have demonstrated that p62 can bind to Keap1, resulting in accumulation of Keap1 in p62 bodies with subsequent autophagic degradation of Keap1. 27,35 Although p62 deficiency results in higher basal Keap1 protein levels, it does not completely prevent, but rather decreases, PA-induced degradation of Keap1. The regulation of Keap1 protein levels is not fully understood and whether additional p62-independent modifications of Keap1 occurs during the lipotoxic process (such as posttranscriptional modifications by phosphorylation, microRNAs, ribosomal occupation, RNA sequestration and so on) remains to be investigated.…”

Depletion of the Ubiquitin-binding Adaptor Molecule SQSTM1/p62 from Macrophages Harboring cftr ΔF508 Mutation Improves the Delivery of Burkholderia cenocepacia to the Autophagic Machinery

“…Successful knockdown was confirmed by immunoblot analysis for each experiment. The DsRed-p62 plasmid was obtained from Addgene (32) and was nucleofected into primary macrophages using a Lonza nucleofection kit and Amaxa equipment. The plasmid was nucleofected 24 h before the infection.…”

p62/SQSTM1 at the interface of aging, autophagy, and disease

“…Alternatively, posttranslational modifications of either Keap1 or p62/SQSTM1 itself may affect binding between the two proteins and provide a physiological mechanism for this alternative NFE2L2 activation. p62/SQSTM1 brings Keap1 into sequestosomes, effectively impairing its ability to bind NFE2L2 (Lau et al 2010), and Keap1 presence into sequestosomes seems to facilitate their clearance through autophagy (Fan et al 2010), which would further reduce Keap1 levels and its ability to bind NFE2L2. p62/SQSTM1 relevance in aging and age-related pathologies p62/SQSTM1 is involved in a variety of cellular processes that are relevant for aging, including protein degradation (Pankiv et al 2007;Seibenhener et al 2004), mitophagy (Narendra et al 2010a;Geisler et al 2010a;Tang et al 2011), oxidative stress response (Jain et al 2010;Komatsu et al 2010;Lau et al 2010), metabolism regulation (Moscat and Diaz-Meco 2011), and inflammation (Lee et al 2012).…”